1. Technical Field
The present invention generally relates to a fluorescence polarization immunoassay, and reagents useful therein, for amphetamine and d-methamphetamine. It provides a preincubation step which is effective to eliminate cross reactivity to .beta.-hydroxyamines. In addition, it relates to the elimination of potential flu rescence interference by riboflavin and potential interference by endogeneous tyramine. Further, the particular methods of chemically synthesizing the novel chemical reagents employed in the novel fluorescence polarization immunoassay of the instant invention eliminate the necessity of utilizing "controlled substances" as starting materials and, thus, eliminate the significant time, effort, and expense which is necessary in order to comply with requirements of the United States Drug Enforcement Agency (USDEA).
2. Background Art
Amphetamine and methamphetamine, the structural chemical formulas of which are presented below, are sympathomimetic phenethylamine derivatives having central nervous system stimulant activity. ##STR1##
These drugs have been used for the treatment of obesity, narcolepsy, and hypotension. However, excessive or prolonged use of these drugs may lead to tolerance and physical dependence. Because of their stimulant effects, the drugs are commonly sold illicitly and abused. Physiological symptoms often associated with very high amounts of ingested amphetamine and methamphetamine include elevated blood pressure, dilated pupils, hyperthermia, convulsions, and acute amphetamine psychosis.
The biological fluid tested most frequently for abuse of amphetamine and methamphetamineis is urine. Other biological fluids have not been extensively investigated for use in assays for the detection of amphetamine and methamphetamine.
In the past, amphetamines have been detected by a number of techniques, including thin layer chromatography (TLC), gas chromatography (GC), and high performance liquid chromatography (HPLC). These methods generally involve chemical extractions of the drugs, complicated procedures requiring highly trained personnel and lengthy assay times. Thin layer chromatography is labor intensive and lacks sensitivity. Gas chromatography and high performance liquid chromatography, each of which is also labor intensive, require highly trained personnel to carry out extractions of the analyte from the biological matrix. In addition, gas chromatography normally requires a derivatization step.
In general, competitive binding immunoassays have provided a preferable alternative to physical methods such as gas chromatography and high performance liquid chromatography.
Fluorescence polarization immunoassay procedures provide a reliable quantitative means for measuring the amount of tracer antibody complex produced in a homogeneous competitive binding assay.
Typically, competitive binding immunoassays are used for measuring ligands in a test sample. Generally, a "ligand" is a substance of biological interest to be determined quantitatively by a competitive binding immunoassay technique. The ligands compete with a labeled reagent, or "ligand analog," or "tracer," for a limited number of receptor binding sites on antibodies specific to the ligand and ligand analog. The concentration of ligand in the sample determines the amount of ligand analog which binds to the antibody. The amount of ligand analog that will bind to the antibody is inversely proportional to the concentration of ligand in the sample, because the ligand and the ligand analog each bind to the antibody in proportion to their respective concentrations.
Fluorescence polarization provides a quantitative or qualitative means for measuring the amount of tracer antibody conjugate produced in a competitive binding immunoassay. Fluorescence polarization techniques are based on the principle that a fluorescent-labeled compound, when excited by plane polarized light, will emit fluorescence having a degree of polarization inversely related to its rate of rotation. Accordingly, when a tracer-antibody conjugate having a fluorescent label is excited with plane polarized light, the emitted light remains highly polarized because the fluorophore is constrained from rotating between the time that light is absorbed and emitted. In contrast, when an unbound tracer is excited by plane-polarized light, its rotation is much faster than the corresponding tracer-antibody conjugate and an excited population of molecules is randomized much more quickly. As a result, the light emitted from the unbound tracer molecules is depolarized.
Such fluorescence polarization techniques have been applied in U.S. Pat. No. 4,420,568 to Wang, et al., which is directed to the use of a triazinylamino-fluorescein moiety as the fluorophore.
An accurate and reliable immunoassay for abuse of amphetamine and d methamphetamine requires that antibody "cross-reactivity" (recognition of compounds other than the desired ligand or ligands) be minimized.
In particular, although both the d- and 1-enantiomers of methamphetamine are USDEA Schedule II controlled substances and, thus, are both considered to have abuse potential, the 1-enantiomer has weaker stimulant activity and is contained in small amounts in some over-the counter medications. It is therefor considered undesirable for an assay which is employed to detect drug abuse to respond to 1-methamphetamine alone in a sample. Therefor, the cross-reactivity for 1-methamphetamine in such an assay should be as close as possible to zero. The combination of novel antisera and tracers employed in the immunoassay of the present invention reduces the cross-reactivity for 1-methamphetamine to below 5.1%.
It is also known that derivatives of .beta.-phenethylamine, particularly .beta.-hydroxyphenethylamine compounds, may be strong interferants in an immunoassay for amphetamine and methamphetamine. One such .beta.-hydroxyphenethylamine, the drug phenylpropanolamine, is found frequently in decongestants sold over the counter. U.S. Pat. No. 3,856,469 discloses removal of .beta.-hydroxyphenethylamine interference from a sample intended for amphetamine or methamphetamine analysis by treating the sample at a pH greater than 8.0 with an amount of aqueous periodate in the presence of ammonium hydroxide. In addition to requiring sample treatment at a basic pH, the aqueous pretreatment in U.S. Pat. No. 3,856,469 is suggested as useful only preceeding sample evaluation by thin layer chromatography and immunoassays by radioimmunoassay, electron spin resonance technique or enzyme technique.
In addition, tyramine, which may be present naturally in a biological sample being analyzed for amphetamine and/or methamphetamine, may also be a strong interferant in an immunoassay for amphetamine and d-methamphetamine. The undesirable result associated with tyramine interference is that false positive results may be obtained. However, the combination of novel antisera and tracers employed in the immunoassay of the present invention significantly improves the selectivity of this immunoassay for amphetamine and d methamphetamine over tyramine in comparison with those methods described in the art in that it maintains the cross-reactivity of the immunoassay for tyramine at about 0.4%.
Further, the particular methods of chemically synthesizing the novel chemical reagents employed in the novel fluorescence polarization immunoassay of the instant invention eliminate the necessity of utilizing "controlled substances" as starting materials and, thus, eliminate the significant time, effort, and expense which is necessary in order to comply with requirements of the federal Drug Enforcement Agency.
Finally, the immunoassay of the present invention provides a more rapid and accurate amphetamine/d methamphetamine assay method than prior art methods because it requires no specimen treatment before analysis and because the assay system has minimal cross-reactivity to 1-methamphetamine or other amphetamine like compounds.
For art relating to the detection of amphetamine and methamphetamine in biological samples, see U.S. Pat. No. 3,996,344 (phenethylamine antigenic conjugates, their preparation, antibodies and use); U.S. Pat. No. 4,016,146 (phenethylamine antigenic conjugates, their preparation, antibodies and use); U.S. Pat. No. 4,041,076 (immunoassay for pharmacologically active phenethylamines); U.S. Pat. No. 4,329,281 (hapten compositions employed in preparing immunogens which are employed in the elicitation of antibodies selective to amphetamine and methamphetamine); U.S. Pat. No. 3,966,764 (ligand determination of spin labeled compounds by receptor displacement-amphetamine analogs); U.S. Pat. No. 4,067,774 (compounds for enzyme amplification assay), U.S. Pat. No. 3,878,187 (polypeptide derivatives of amphetamine and analogs for immunoassays); FEBS LETTERS 36, 3 1973) (radioimmunoassay procedure for measuring amphetamines in urine); Chem. Pharm. Bull. 25(4), 840 (1977) (radioimmunoassay for methamphetamine); Forensic Science International 27, 49 ( 985) (a latex agglutination inhibition reaction test for screening urinary amphetamine); Analytical Biochemistry 161, 117 (1987) (the induction of methamphetamine-specific antibody using biodegradable carboxymethyl-chitin); Journal of Medicinal Chemistry, Vol. 19, No. 1 (1976) (determination of the specificity of an antibody directed against d-(S)-methamphetamine); Clin. Chem. 32/9, 1677 (1986)(a homogeneous fluoroimmunoassay for detecting amphetamines in urine ; Jpn J Legal Med 37(4), 417 (1983) (solid phase microELISA for methamphetamine); Journal of Forensic Sciences, Vol. 32, No. 3, 658 (1987) (histochemical demonstration of methamphetamine by immunocytochemistry); Chem. Pharm. Bull. 25(4), 838 (1977) (preparation of a specific antibody to methamphetamine); Clinical Toxicology, 18(1), 91 (1981) (analysis of amphetamine-related amines by EMIT at different concentrations); Analytical Biochemistry 60, 551 (1974) (radioimmunoassay of 3,4,5 -Trimethoxy phenethylamine (mescaline) and 2,5-Dimethoxy 4-Methylphenyl-isopropylamine); and Clinical Toxicology, 18(3), 299 (1981) (analysis of amphetamine-related amines by RIA).
Accordingly, a need exists for providing a method and reagents for performing a reliable and accurate fluorescence polarization assay for both amphetamine and d-methamphetamine in biological fluids such as urine. The present invention is an advance in the art in that novel reagents specifically useful in fluorescence polarization immunoassays for amphetamine and d-methamphetamine, and a novel combination of such reagents in such immunoassays, are provided.